Nitric oxide, phosphodiesterase inhibition, and adaptation to hypoxic conditions.
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3',5'-Cyclic-GMP Phosphodiesterases↗
Biomedical subjects
M R Wilkins
Publications and source records attributed to M R Wilkins.
What do we want from proteomics in the detection and avoidance of adverse drug reactions.
The incidence of severe adverse drug reactions is approximately 7% in hospital patients. In many cases the adverse event is difficult to predict or even explain on the basis of the known pharmacology of the causative agent. It is not infrequent in the context of multiple drug therapy, which complicates identification of the culprit. This can present a major problem in the management of chronic diseases such as tuberculosis or epilepsy. Pharmacogenetics offers one approach to reducing the incidence of drug-induced adverse reactions but has recognised limitations as a predictive tool and little role in diagnosis. Proteomics may have some predictive value but is likely to be of greater use in diagnosis-for example by recognising a drug signature in an accessible tissue. This may be possible on a blood sample or biopsy taken at presentation. Alternatively an in vitro assay that replaced rechallenging the patient with a drug would be helpful. The goal is to identify the causative drug permitting resumption of treatment with a safer alternative.
Drug-Related Side Effects and Adverse Reactions↗
In vivo production of interferon beta by human Tenon's fibroblasts; a possible mediator for the development of chronic conjunctival inflammation.
BACKGROUND: Chronic inflammation may develop from failure of the immune system to deactivate itself during resolution of the wound healing response, and is recognised as a major risk factor for trabeculectomy failure. Fibroblast/T cell interactions may contribute to aggressive scarring. Our previous research showed that in vitro human Tenon's fibroblast produced interferon beta was responsible for preventing T cell apoptosis, suggesting that this interaction could contribute to the development of chronic inflammation. METHODS: Immunohistological techniques were used to investigate the in vivo components of this particular fibroblast/T cell interaction in conjunctival biopsies from glaucoma patients undergoing filtration surgery. RESULTS: Fibroblast produced interferon beta and T lymphocytes were identified in human conjunctiva. CONCLUSION: The components of fibroblast mediated prevention of T cell apoptosis were identified in vivo, suggesting that the development of this interaction is possible and that it may contribute to the development of chronic inflammation and excessive scarring.
Aged↗
Natriuretic peptide receptors and the heart.
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Atrial Natriuretic Factor↗
Data standardisation in GlycoSuiteDB.
GlycoSuiteDB, a database of glycan structures, has been constructed with an emphasis on quality, consistency and data integrity. Importance has been placed on making the database a reliable and useful resource for all researchers. This database can help researchers to identify what glycan structures are known to be attached to certain glycoproteins, as well as more generally identifying what types of glycan structures are associated with different states, for example, different species, tissues and diseases. To achieve this, a major effort has gone into data standardisation. Many rules and standards have been adopted, especially for representing glycan structure and biological source information. This paper describes some of the challenges faced during the continuous development of GlycoSuiteDB.
Animals↗
Sildenafil inhibits hypoxia-induced pulmonary hypertension.
BACKGROUND: This study investigated the effect of the phosphodiesterase 5 inhibitor sildenafil on the pulmonary vascular response to hypoxia in humans and mice. METHODS AND RESULTS: In a randomized, double-blind study, sildenafil 100 mg or placebo was given orally to 10 healthy volunteers 1 hour before breathing 11% O(2) for 30 minutes. Pulmonary artery pressure (PAP) was measured with an indwelling right heart catheter. The acute 56% increase in mean PAP produced by hypoxia during placebo treatment (mean PAP [mean+/-SD mm Hg]: normoxia 16.0+/-2.1 versus hypoxia 25.0+/-4.8) was almost abolished by sildenafil (normoxia 16.0+/-2.1 versus hypoxia 18.0+/-3.6), with no significant effect on systemic blood pressure. In the isolated perfused lung of wild-type and endothelial nitric oxide synthase (eNOS)-deficient mice, sildenafil markedly blunted acute hypoxic pulmonary vasoconstriction. Wild-type mice dosed orally with the drug (25 mg. kg(-1). d(-1)) throughout 3 weeks of exposure to hypoxia (10% O(2)) exhibited a significant reduction in right ventricular systolic pressure (placebo versus sildenafil: 43.3+/-9.9 versus 29.9+/-9.7 mm Hg, P<0.05) coupled with a small reduction in right ventricular hypertrophy and inhibition of pulmonary vascular remodeling. In eNOS mutant mice, sildenafil attenuated the increase in right ventricular systolic pressure but without a significant effect on right ventricular hypertrophy or vascular remodeling. CONCLUSIONS: Sildenafil attenuates hypoxia-induced pulmonary hypertension in humans and mice and offers a novel approach to the treatment of this condition. The eNOS-NO-cGMP pathway contributes to the response to sildenafil, but other biochemical sources of cGMP also play a role. Sildenafil has beneficial pulmonary hemodynamic effects even when eNOS activity is impaired.
Adolescent↗
Right ventricular hypertrophy secondary to pulmonary hypertension is linked to rat chromosome 17: evaluation of cardiac ryanodine Ryr2 receptor as a candidate.
BACKGROUND: Fischer 344 (F344) rats are relatively resistant to hypoxia-induced right ventricular (RV) hypertrophy compared with the Wistar-Kyoto (WKY) strain. These 2 strains were used to examine the genetic basis for the differential response. METHODS AND RESULTS: Male F(2) offspring from an F344xWKY intercross were exposed to hypoxia (10% O(2)) for 3 weeks, and pulmonary artery pressure and cardiac chamber weights were measured. Genomic DNA was screened by use of polymorphic microsatellite markers across the whole genome (excluding the sex chromosomes). A quantitative trait locus (QTL) for RV weight was identified on rat chromosome 17 (lod score 6.5) that accounted for 22% of the total variance of RV weight in the F(2) population and was independent of pulmonary artery pressure. The peak was centered over marker D17Rat41, close to Chrm3, with a 1-lod support interval of 5 cM. Comparison of homologous regions in mice and humans suggested that Ryr2, the cardiac isoform of the ryanodine receptor, colocalizes with our QTL. A panel of somatic cell hybrids and fluorescence in situ hybridization mapped Ryr2 close to the gene Chrm3 within our QTL. [(3)H]Ryanodine binding to cardiac membranes from the parental strains showed a 21% reduction in B(max) in the WKY compared with the F344 strain, with no difference in K:(d). CONCLUSIONS: These data provide the first demonstration of a QTL linked to the RV response to hypoxia-induced pulmonary hypertension. The Ryr2 receptor gene lies within this QTL and merits further investigation as a candidate for this differential RV response.
Animals↗
GlycoSuiteDB: a new curated relational database of glycoprotein glycan structures and their biological sources.
GlycoSuiteDB is a relational database that curates information from the scientific literature on glyco-protein derived glycan structures, their biological sources, the references in which the glycan was described and the methods used to determine the glycan structure. To date, the database includes most published O:-linked oligosaccharides from the last 50 years and most N:-linked oligosaccharides that were published in the 1990s. For each structure, information is available concerning the glycan type, linkage and anomeric configuration, mass and composition. Detailed information is also provided on native and recombinant sources, including tissue and/or cell type, cell line, strain and disease state. Where known, the proteins to which the glycan structures are attached are reported, and cross-references to the SWISS-PROT/TrEMBL protein sequence databases are given if applicable. The GlycoSuiteDB annotations include literature references which are linked to PubMed, and detailed information on the methods used to determine each glycan structure are noted to help the user assess the quality of the structural assignment. GlycoSuiteDB has a user-friendly web interface which allows the researcher to query the database using mono-isotopic or average mass, monosaccharide composition, glycosylation linkages (e.g. N:- or O:-linked), reducing terminal sugar, attached protein, taxonomy, tissue or cell type and GlycoSuiteDB accession number. Advanced queries using combinations of these parameters are also possible. GlycoSuiteDB can be accessed on the web at http://www.glycosuite.com.
Animals↗
Strategy for protein isoform identification from expressed sequence tags and its application to peptide mass fingerprinting.
Expressed Sequence Tags (ESTs) are an invaluable resource for protein identification and characterisation in proteomics. They allow proteins to be identified in the absence of genome sequence data. When EST sequences are used for protein identification, they are usually first processed into contigs to reduce redundancy and generate longer sequences from the overlapping ESTs. However, the process of generating contigs may accidentally group biologically meaningful isoforms together. Here we report means of discovering isoforms in EST sequences and how to use this information in the framework of protein identification and characterisation with peptide mass fingerprinting. We illustrate our strategies with examples from the dbEST database as well as protein isoforms from two-dimensional polyacrylamide gels.
Amino Acid Sequence↗
Progress in, and future prospects for, the treatment of primary pulmonary hypertension.
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Administration, Oral↗
Characterization of adenylyl cyclase isoforms in rat peripheral pulmonary arteries.
Activation of adenylyl cyclase (AC), of which there are 10 diversely regulated isoforms, is important in regulating pulmonary vascular tone and remodeling. Immunohistochemistry in rat lungs demonstrated that AC2, AC3, and AC5/6 predominated in vascular and bronchial smooth muscle. Isoforms 1, 4, 7, and 8 localized to the bronchial epithelium. Exposure of animals to hypoxia did not change the pattern of isoform expression. RT-PCR confirmed mRNA expression of AC2, AC3, AC5, and AC6 and demonstrated AC7 and AC8 transcripts in smooth muscle. Western blotting confirmed the presence of AC2, AC3, and AC5/6 proteins. Functional studies provided evidence of cAMP regulation by Ca(2+) and protein kinase C-activated but not G(i)-inhibited pathways, supporting a role for AC2 and a Ca(2+)-stimulated isoform, AC8. However, NKH-477, an AC5-selective activator, was more potent than forskolin in elevating cAMP and inhibiting serum-stimulated [(3)H]thymidine incorporation, supporting the presence of AC5. These studies demonstrate differential expression of AC isoforms in rat lungs and provide evidence that AC2, AC5, and AC8 are functionally important in cAMP regulation and growth pathways in pulmonary artery myocytes.
Adenylyl Cyclases↗
A gene for primary pulmonary hypertension.
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Bone Morphogenetic Protein Receptors, Type II↗
Proteomics: capacity versus utility.
Until recently scientists studied genes or proteins one at a time. With improvements in technology, new tools have become available to study the complex interactions that occur in biological systems. Global studies are required to do this, and these will involve genomic and proteomic approaches. High-throughput methods are necessary in each case because the number of genes and proteins in even the simplest of organisms are immense. In the developmental phase of genomics, the emphasis was on the generation and assembly of large amounts of nucleic acid sequence data. Proteomics is currently in a phase of technological development and establishment, and demonstrating the capacity for high throughput is a major challenge. However, funding bodies (both in the public and private sector) are increasingly focused on the usefulness of this capacity. Here we review the current state of proteome research in terms of capacity and utility.
Animals↗
Post-operative 5-fluorouracil for glaucoma surgery.
BACKGROUND: Trabeculectomy is performed as a treatment for many types of glaucoma in an attempt to lower the intra-ocular pressure. The surgery involves creating a channel through the sclera, through which intra-ocular fluid can leave the eye. If scar tissue blocks the exit of the surgically created channel intra-ocular pressure rises and the operation fails. Antimetabolites are used to inhibit wound healing to prevent the conjunctiva scarring down on to the sclera. The principal antimetabolites used are 5-Fluorouracil and Mitomycin C. Both may be applied on a sponge between the conjunctiva and sclera at the beginning of surgery. 5-Fluorouracil may also be given as one or more injections under the conjunctiva after the surgery. OBJECTIVES: The objective of this review is to assess the effects of post-operative injections of 5-Fluorouracil in eyes of people undergoing surgery for glaucoma. SEARCH STRATEGY: We searched the Cochrane Eyes and Vision Group specialised register, The Cochrane Controlled Trials Register (CENTRAL), MEDLINE and EMBASE. We searched the reference lists of relevant articles for additional trials, and we used the Science Citation Index to search for articles that cited the included studies. We contacted investigators and experts for details of additional relevant trials. SELECTION CRITERIA: We included randomised trials of post-operative 5-Fluorouracil injections compared to placebo injections or no injections in trabeculectomy for glaucoma. DATA COLLECTION AND ANALYSIS: We independently assessed trial quality and extracted data. We contacted trial investigators for missing information. Data were summarised using relative risk, Peto odds ratio and weighted mean difference as appropriate. MAIN RESULTS: Eight trials, which randomised a total of 536 participants, are included in the review. As far as can be determined from the trial reports, the methodological quality of the trials was not high. 5-Fluorouracil when delivered by post-operative injection appears to be effective in reducing the likelihood of surgical failure of trabeculectomy both in eyes at high risk of failure and those undergoing surgery for the first time. Complications are more common after 5-Fluorouracil injections, especially early and late wound leaks and temporary damage to the ocular surface. Whilst no evidence was found of an increased risk of serious sight threatening complications, people undergoing initial trabeculectomy had a substantially increased risk of ocular hypotony. None of the trials reported on the participants' perspective of care. REVIEWER'S CONCLUSIONS: This treatment is now rarely used on the basis of a planned series of post-operative injections.This presumably reflects an aspect of the treatment that is unacceptable to both patients and doctors. None of the trials reported on the participants' perspective of care which constitutes a serious omission for an invasive treatment such as this. Increasingly, injections are used on an ad hoc basis. The effectiveness of this strategy is the subject of a future systematic review.
Antimetabolites↗
Automatic poisson peak harvesting for high throughput protein identification.
High throughput identification of proteins by peptide mass fingerprinting requires an efficient means of picking peaks from mass spectra. Here, we report the development of a peak harvester to automatically pick monoisotopic peaks from spectra generated on matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometers. The peak harvester uses advanced mathematical morphology and watershed algorithms to first process spectra to stick representations. Subsequently, Poisson modelling is applied to determine which peak in an isotopically resolved group represents the monoisotopic mass of a peptide. We illustrate the features of the peak harvester with mass spectra of standard peptides, digests of gel-separated bovine serum albumin, and with Escherictia coli proteins prepared by two-dimensional polyacrylamide gel electrophoresis. In all cases, the peak harvester proved effective in its ability to pick similar monoisotopic peaks as an experienced human operator, and also proved effective in the identification of monoisotopic masses in cases where isotopic distributions of peptides were overlapping. The peak harvester can be operated in an interactive mode, or can be completely automated and linked through to peptide mass fingerprinting protein identification tools to achieve high throughput automated protein identification.
Animals↗
Serotype classification and characterisation of the rotavirus SA11 VP6 protein using mass spectrometry and two-dimensional gel electrophoresis.
VP6, which makes up the inner capsid of rotavirus, is the major structural protein of this virus. Whilst VP6 has been sequenced at the DNA level in several rotavirus strains, there has been less effort to characterise the protein at the amino acid level. This paper reports the use of peptide mass fingerprinting and post-source decay fragmentation studies using MALDI-TOF and electrospray ionisation mass spectrometry to identify and characterise, in detail, the VP6 protein. We show that mass spectrometric analysis of VP6 peptides successfully distinguished SA11 from other rotavirus serotypes, and identify unique peptides that can be used for serotypic differentiation. For VP6 characterisation, the ExPASy FindMod tool was used to predict post-translational modifications on the protein. Analysis of trypsin and AspN digests predicted that the N-terminal methionine of VP6 was acetylated and this was confirmed using post source decay and electrospray ionisation mass spectrometry-mass spectrometry. An asparagine residue (aa107), which is followed by a glycine residue, was shown to undergo partial deamidation to aspartic acid. VP6 has two additional asparagine-glycine sequences and, in this sequence context, asparagine is known to be particularly susceptible to deamidation. Two-dimensional gel electrophoresis revealed a complex series of VP6 isoforms with an apparent molecular mass of approximately 45,000 Da and a pI ranging from 5.25 to 5.8. This pattern could partly be explained by the potential for deamidation at several sites within the protein.
Acetylation↗
Drug action at the 5-HT(1A) receptor in vivo: autoreceptor and postsynaptic receptor occupancy examined with PET and [carbonyl-(11)C]WAY-100635.
Serotonin(1A) (5-HT(1A)) receptors have been implicated in the pathophysiology and treatment of anxiety and depression and are a target for novel drug development. In this qualitative study, positron emission tomography (PET) and [carbonyl-(11)C]WAY-100635 were used to assess 5-HT(1A) autoreceptor and postsynaptic receptor occupancy in man in vivo by five different compounds with nanomolar affinity for this site. Occupancy by pindolol, penbutolol, buspirone, EMD 68843, and S 15535 was compared to test-retest data from 10 healthy volunteers. All drugs, apart from buspirone, displayed occupancy at the 5-HT(1A) receptor site. Pindolol demonstrated a preferential occupancy at the autoreceptor compared to the postsynaptic receptor over a plasma range of about 10-20 ng/mL. Differential occupancy may be an important component of novel drug action. The level of autoreceptor or postsynaptic occupancy needed to achieve significant physiological effects is not known, although it is of note that none of the drugs in this study achieved occupancies beyond 60%. Overall this study demonstrates the utility of PET in aiding novel drug development.
Adult↗
Sponge delivery variables and tissue levels of 5-fluorouracil.
AIM: To study how the delivery of 5-fluorouracil (5-FU) to ocular tissues is affected by altering delivery variables. METHOD: Sponge(s) soaked in radiolabelled 5-FU were placed between the conjunctiva and sclera of pig eyes. Application time, sponge size, sponge make (Altomed, Weck, Merocel), and 5-FU concentration were varied. Conjunctival and scleral tissue levels were determined in samples taken from the application site. RESULTS: Dose-response curves for scleral and conjunctival 5-FU levels against application time showed increasing tissue levels that reached a plateau after 2-3 minutes. Application beyond 3 minutes did not increase tissue levels. There was no difference in tissue levels between 7x4 and 3. 5x2 mm sponges. Altomed sponges produced 5-FU tissue levels that were twice as high as those obtained with Weck-cell (p<0.01) or Merocel (p<0.02) sponges. Changing the 5-FU concentration from 25 mg/ml to 6.25 mg/ml reduced the conjunctival concentration by a factor of 3.5 (p<0.003). CONCLUSION: Application time up to 3 minutes, sponge make, and 5-FU concentration can have a large effect on the tissue delivery of 5-FU. Application time beyond 3 minutes, using 3.5x2 mm or 7x4 mm sponges, and replacing sponges every minute did not have a significant effect on tissue levels. This study models the effect that different variables can have on the ocular tissue levels of an antimetabolite applied intraoperatively.
Administration, Topical↗